Project Summary Suppressor or regulatory T cells (Tregs) play a key role in maintaining T cell homeostasis and preventing autoimmunity. In addition, inappropriate expansion of Tregs can play an important role in suppressing immune-mediated anti-tumor responses. Thus, Tregs play an important role in both autoimmune disease and cancer. However, the molecular mechanisms that regulate Treg development remain poorly characterized. We currently know that signals emanating from the TCR, the co-stimulatory molecule CD28, and the family of common gamma chain (&#947;c)-dependent cytokine receptors all play a role in the development and homeostasis of CD4+Foxp3+ regulatory T cells. However, how these signals interact with one another to induce Treg differentiation and selection of the Treg TCR repertoire remains unclear. Importantly, recent studies have identified a distinct population of CD4+Foxp3-CD25+ thymocytes that are direct progenitors of CD4+Foxp3+ Tregs;these CD4+Foxp3- progenitors can be rapidly converted into CD4+Foxp3+ Tregs upon IL2 stimulation alone. Moreover, using mice that express a constitutively active STAT5 transgene (STAT5b-CA) in developing thymocytes we have demonstrated that ectopic STAT5 signals are sufficient to convert thymocytes expressing a TCR that is normally destined to become a na[unreadable]ve T cell into the Treg lineage. Based on these preliminary findings we propose a two-step model for Treg development. The first step involves a TCR/CD28-dependent step that results in the generation of the Treg progenitor. The second step involves differentiation of these primed Treg progenitors into mature Foxp3+ Tregs if they receive the appropriate cytokine-derived signals. Under physiological circumstances this cytokine appears to be IL2. We, and others, have found that IL7 and IL15 can substitute for IL2 in IL2-/- mice in driving Treg differentiation (albeit rather poorly), but this does not occur in WT mice as IL2 acts to suppress expression of those receptors on Tregs. Thus IL2 is the key cytokine responsible for completing Treg differentiation in the thymus. Based on these findings, we propose that restricted localization of IL2 in the thymus plays an important role in shaping the repertoire of TCRs expressed by Tregs. Thus, the aims of this grant are to (i) determine how TCR/CD28-dependent signals initiate Treg progenitor differentiation (ii) determine how IL2/STAT5-dependent signals drive conversion of Treg progenitors into mature Tregs, and (iii) identify which cells produce the IL2 in the thymus that is required for Treg differentiation. These studies will illuminate the process by which STAT5 directs regulatory T cell differentiation and provide us with a better understanding of how the Treg repertoire is matched with that of autoreactive T cells. Furthermore, they should provide insights into molecular mechanisms that will allow for the manipulation of Treg numbers and function and thereby prove useful for designing novel approaches aimed at preventing or suppressing autoimmune diseases.